Many biomolecules, including proteins and peptides, hold potential as reagents for use in diagnosis and therapy of human conditions and diseases. As most biomolecules do not, by themselves, have properties to make them useful as diagnostic and/or therapeutic reagents, biomolecules of interest are often chemically modified to achieve this. However, one very important criterion must be applied when chemically modifying biomolecules. That criterion is that the modification does not alter the biological property that is important (e.g. cancer cell targeting) in the use of that particular biomolecule. This criterion makes it imperative that site-selective (where possible) and minimal modification of the biomolecule be conducted.
Modification of a targeting biomolecule with an effector agent, such as a radionuclide, can provide valuable new tools for diagnosis and therapy of human and animal diseases or conditions. Coupling of a radionuclide to the biomolecule results in the desired diagnostic effect of providing photons that can be measured or imaged externally to show the localization of the radiolabeled biomolecule, or it may provide the desired therapeutic effect of causing damage to cells or tissues that are targeted by the biomolecule. Biomolecules labeled with photon emitting radionuclides can be used for the diagnosis of a number of human conditions (i.e. extent of myocardial infarcts, presence of cancer, etc.). For example, technetium-99m labeled antibodies can be used to diagnose cancer (Granowska et al. Eur. J. Nucl. Med. 20, 483-489, 1993; Lamki et al. Cancer Res. 50, 904s-908s, 1990; Goldenberg et al. Cancer Res. 50, 909s-921s, 1990); iodine-123 labeled fatty acids can be used to evaluate myocardial perfusion (Corbett J. Nucl. Med. 35, 32s-37s, 1994; Hansen J. Nucl. Med. 35, 38s-42s, 1994; Knapp et al. J. Nucl. Med. 36, 1022-1030, 1995); and fluorine-18 labeled fluorodeoxyglucose can be used to evaluate a variety of functions of the brain (Posner et al., Science 240, 1627-1631, 1988). Biomolecules labeled with particle emissions (e.g. beta, positron, alpha, Auger electrons) can potentially be used for targeted radiotherapy of human disease such as cancer. For example, a large number of monoclonal antibodies (Behr et al. J. Nucl. Med. 38, 858-870, 1997; Divgi et al. J. Nucl. Med. 36, 586-592, 1995; DeNardo et al. Anticancer Res. 17, 1735-1744, 1997) and peptides (Zamora et al. Int. J. Cancer 65, 214-220, 1996; Stolz et al. Digestion 57, 17-21, 1996; Bender et al. Anticancer Res. 17, 1705-1712, 1997) labeled with therapeutic radionuclides such as iodine-131, yttrium-90 and Re-188 are being investigated as new reagents for cancer therapy. Thus, an important modification that can be carried out is to attach a functional moiety to the biomolecule which binds or bonds with a radionuclide. For small (i.e. <2000 Da molecular weight) biomolecules, usually only one radionuclide binding/bonding moiety is site-selectively attached to cause minimal perturbation in its desired biological properties. Larger biomolecules, such as peptides and proteins, may be conjugated with more than one radionuclide binding/bonding moiety before loss of the desired biological properties, but these molecules generally retain more of their desired biological properties when minimal number of conjugations are obtained.
Modification of biomolecules with an “affinity ligand” is also important as it provides a means of coupling two entities together for a variety of in vitro and in vivo applications. By their nature, affinity ligands come in pairs. The preferred affinity ligands used for coupling to the biomolecule must have a high enough binding constant (e.g. 106 M−1 or greater) with a second compound to allow the two coupled entities to remain together for a period of time. An example of an affinity ligand pair is a monoclonal antibody and its hapten. The affinity ligand pairs of biotin/avidin and biotin/streptavidin are often used with biomolecules. The very strong interaction (i.e. K=1013-1015 M−1) of biotin with the proteins avidin and streptavidin (Green, Methods Enzymol. 184, 51-67, 1990; Green, Adv. Prot. Chem. 29, 85-133, 1975) provides a foundation for their use in a large number of applications, both for in vitro and in vivo uses. While the proteins avidin and streptavidin are sometimes conjugated with biomolecules, conjugation of biotin introduces less perturbation of the biomolecule, and more than one biotin molecule can be conjugated with minimal affect on the biomolecule. Therefore, the preferred affinity label is biotin or a derivative thereof, and the examples herein are reflective of this preference. As with the radionuclide binding/bonding moiety, it is important to minimize the number of affinity ligands (e.g. biotin conjugates) attached to a biomolecule to retain the desired biological properties.
Modification of the biomolecule by attachment (conjugation) of another molecule to a particular reactive functional group (e.g. amine, sulfhydryl, aldehyde, ketone) precludes attachment of a second molecule to that group. Thus, if attachment of more than one type of molecule to a biomolecule is desired (to impart two functions), the attachment must be made at a second site using currently available reagents. Since in some applications, it is desirable to have both an affinity ligand and an effector agent (e.g. a moiety that binds/bonds with a radionuclide), site-selective conjugation is precluded. Further, modification of biomolecules that are not made in a site-selective manner (e.g. reaction with surface amine groups in proteins) are limited due to the fact that two different sites are modified. Additionally, modification of larger biomolecules (e.g. proteins) in two subsequent steps can result in a heterogeneous population of modified biomolecules in which molecules that contain the second conjugated species may have less of the desired biological properties (i.e. tumor targeting) than those that do not contain the second conjugate. This can result in a subgroup of biomolecules containing both conjugated species that do not have the properties desired. To circumvent these problems, the affinity ligand (e.g. biotin moiety) and an effector agent (e.g. radionuclide binding/bonding moiety with or without the radionuclide) can be coupled together through trifunctional cross-linking reagent to form a new type of reagent. With the use of this new class of reagents, an equal number of affinity ligands and radionuclide binding/bonding moieties will be conjugated to the biomolecule. With a combined affinity ligand and radiolabeling compound, site specific addition of both reagents can be made, and minimization of the number of conjugates to the biomolecule can be attained. Linking an affinity ligand such as biotin to a fluorescent moiety which is further attached to an oligosaccharide is described in Varki et al., WO 94/28008. The issue of attaching an affinity ligand to cytotoxic agent or an agent which can convert a prodrug to an active drug, and where either of these are further attached to a targeting molecule, is addressed in Nilsson et al., U.S. patent application Ser. No. 08/090 047. However, none of these publications neither alone or in combination describe or indicate the present innovation. The issue of combining an affinity reagent and effector agent on one molecule to achieve minimal modification of biomolecules is not unique to biotin (as the affinity ligand) or radionuclide binding/bonding moieties (the effector agent), and is not limited to only one affinity ligand and one effector ligand per molecule. Combinations of more than one affinity ligand and/or more than one affinity ligand per molecule may be advantageous for certain applications.
The radiolabeled and affinity ligand conjugated biomolecule products obtained from this invention are useful in many in vitro and in vivo applications. A preferred application, where the biomolecule is a tumor binding monoclonal antibody, toxin conjugate, or enzyme conjugate, the affinity ligand is biotin or a derivative thereof, and the radionuclide is a diagnostic or therapeutic radionuclide used in a patient cancer treatment protocol, is to use a biotin binding (e.g. avidin coated) column for extracorporeal immunoabsorptive removal of a radiolabeled antibody conjugate from a patient's blood. Extracorporeal removal of the radiolabeled antibody, toxin conjugate, or enzyme conjugate limits the toxic effects of the radioactivity, toxin, or enzyme to specifically targeted tissues, minimizing the exposure time and interaction with non-target tissues. Importantly, to be effective, medical agents (e.g. biomolecules) must exert their pharmacological action on a particular target tissue or group of target cells. Targeting of such agents is most often carried out by systemic administration (i.e. intravenous injection) which means that they will be transported through the blood and lymph system to most parts of the body. This transportation, or circulation, of the medical agent throughout the body can result in undesirable toxic side effects in tissues or organs other than those where the effect of the agents is beneficial to the patient.
Specific tissue or organ localization of a medical agent is a very important factor in its effective application. Lack of specific tissue localization is of particular importance in the treatment with medical agents where the desired effect is to kill certain types of cells such as in the treatment of cancer. In order to increase the specificity and thereby make the cancer therapy more effective, tumor marker specific targeting agents such as cancer cell binding monoclonal antibodies have been used as carriers for various cell toxic agents (immunoconjugates) such as, but not limited to, radionuclides, cytotoxins, and enzymes used in prodrug protocols (Meyer et al., Bioconjugate Chem. 6, 440-446, 1995; Houba et al., Bioconjugate Chem. 7, 606-611, 1996; Blakey et al., Cancer Res. 56, 3287-3292, 1996). Although, monoclonal antibodies are selectively bound with tumor cells over non-tumor cells, an initial high concentration of the toxic immunoconjugate is required to optimize binding of a particular agent with tumors in a patient. While required for optimal therapy of the cancer, the high concentration of cytotoxic material in blood and non-target tissues causes undesirable side-effects on sensitive and vital tissues like the bone marrow. Various methods have been proposed to rapidly clear these agents from blood circulation after that the tumor has received a maximum dose of the immunoconjugate. Some blood clearance methods involve the enhancement of the bodies own clearing mechanism through the formation of various types of immune complexes. Similarly, blood clearance can be obtained by using molecules that bind with the immunoconjugate, such as monoclonal antibodies (Klibanov et al., J. Nucl. Med. 29, 1951-1956, 1988; Marshall et al., Br. J. Cancer 69, 502-507, 1994; Sharkey et al. Bioconjugate Chem. 8, 595-604, 1997), (strept)avidin (Sinitsyn et al., J. Nucl. Med. 30, 66-69, 1989; Marshall et al., Br. J. Cancer 71, 18-24, 1995), or biotin containing compounds which also contain sugar moieties recognized by the asialoglycoprotein receptor on liver cells (Ashwell and Morell, Adv. Enzymol. 41, 99-128, 1974). Other methods involve means of removing the circulating immunoconjugates through extracorporeal methods (see review article by Schriber G. J. & Kerr D E, Current Medicinal Chemistry, 1995, Vol. 2, pp 616-629).
The extracorporeal techniques used to clear a medical agent from blood circulation is particularly attractive. Extracorporeal devices for this application have been described (Henry C A, 1991, Vol. 18, pp. 565; Hofheinz D et al, Proc. Am. Assoc. Cancer Res. 1987 Vol. 28, pp. 391; Lear J L, et al. Radiology 1991, Vol. 179, pp. 509-512; Johnson T K, et al. Antibody Immunoconj. Radiopharm. 1991, Vol. 4, pp. 509; Dienhart D G, et al. Antibody Immunoconj. Radiopharm. 1991, Vol. 7, pp. 225; DeNardo G L, et al. J. Nucl. Med. 1993, Vol. 34, pp. 1020-1027; DeNardo G L, et al. J. Nucl. Med. 1992b, Vol. 33, pp. 863-864; DeNardo S. J., et. al. J. Nucl. Med. 1992a, Vol. 33, pp. 862-863. U.S. Pat. No. 5,474,772; Australian patent 638061, EPO 90 914303.4 of Maddock, describe these methods.
To make the blood clearance more efficient and to enable processing of whole blood, rather than blood plasma, the medical agent (e.g. tumor specific monoclonal antibody carrying cell killing agents or radionuclides for tumor localization) have been biotinylated and cleared with the use of an affinity (e.g. biotin-binding) column. A number of publications provide data which show that this technique is both efficient and practical for the clearance of biotinylated and radionuclide labeled tumor specific antibodies (Norrgren K, et al. Antibody Immunoconj Radiopharm 1991, Vol. 4, pp. 54; Norrgren K, et. al. J. Nucl. Med. 1993, Vol. 34, pp. 448-454 Garkavij M, et. al. Acta Oncologica 1996, Vol. 53, pp. 309-312; Garkavij M, et. al. J. Nucl. Med. 1997, Vol. 38, pp. 895-901). U.S. patent application Ser. No. 08/090,047, EPO 92 903 020.3 of Nilsson and Ser. No. 08/434,889 of Maddock describe these applications.